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Protocol
  • © M. Rafiq Islam
  • , et al.
  • 2022

Long Term Storage, Recycling and Simplified Calcium Phosphate Transfection Protocols for Cell Cultures

  • M. Rafiq Islam 1
  • Huixun Du 2
  • Huixun Du 2
  • Heejae Nicole Shim 3
  • Bintha Bhandari 1
  • 1 - Laboratory of Biochemistry - Department of Natural Sciences - Northwest Missouri State University -Maryville MO 64468
  • 2 - The Buck Institute for Research on Aging CA
  • 3 - Department of Biochemistry - University of California-Berkley

Jun 16, 2022

Abstract

Long term storage of cells is vital for use in the future experiments. Current methods of storing cells under liquid nitrogen vapor is costly, and requires regular monitoring and filling of the storage tank. We stored several cell lines in -80oC freezer over a decade and revived them successfully. Storing cells in a -80oC freezer does not require regular monitoring, is free of extra cost for N2 and almost hassle-free.  Single use of tissue culture dishes for cell maintenance, besides generating plastic wastes, becomes costly when working with multiple cell types. Repeated use of tissue culture dishes for cell maintenance reduces waste and cost significantly. A modified method for transfection using calcium phosphate will save sample preparation time and increase efficiency. Here we describe modified methods for cell storage, recycling culture dishes and a simple transfection using calcium phosphate. 


Introduction

Long term storage of cells for use in the future is vital in many fields of research and application. The current gold standard is to store cells under liquid N2 vapor. However, besides cost, it often requires regular monitoring and filling of the storage tank with liquid N2. If not done properly or if the tank is depleted of liquid N2 due to negligence or alarm failure of the storage tank, the result is often loss of important cells. We stored a number of cell lines including HEK293T, HepG2, HCT, HeLa, MEF, and mouse Leydig cells in -80oC freezers from 2 years to over 10 years, and revived each successfully. Storage in a -80oC freezer does not require regular monitoring, is free of extra cost for liquid N2 and almost hassle-free. 

One-time use of tissue/cell culture dishes (TCD) is pretty standard for cell maintenance and experiments. Besides generating plastic wastes, TCDs are relatively expensive, especially for low budget laboratories. The cost increases significantly when working with multiple cell lines. We reused TCDs for cell maintenance for several cell lines for years. Multiple-time use of TCDs for cell maintenance will reduce the cost and plastic waste generated.

A number of methods have been developed for introduction of foreign DNA into mammalian cells. Of these, the calcium phosphate (CaPh) method is widely used as it is inexpensive, and compared to the DEAE-dextran method, can be used for both transient and stable transfections. The current CaPh method1-4 calls for simultaneous bubbling of each DNA sample mixed with Ca+2 while phosphate-buffer is added. The separate bubbling of each DNA sample is time consuming, which multiplies exponentially with large sample numbers. The method described here is what we have used for years. It will save time for sample preparation and also increase transfection efficiency.


Reagents and Equipment

Reagents

DMEM (Dulbecco’s modification of Eagle’s Medium with glucose, glutamine and pyruvate), MEM (minimum Essential medium, with Earbalanced salt solution) or RPMI 1640 medium containing antibiotics penicillin-streptomycin (PS, 5000 u/mL pencillin and 5 mg streptomycin) with or without heat-inactivated 10% fetal bovine serum (FBS), trypsin-EDTA (TE, 0.05% trypsin and 0.02%EDTA), 2x Hepes (42 mM Hepes, 274 mM NaCl, 10 mM KCl, 1.8 mM Na2HPO4, pH 7.1), 2M CaCl2. DMSO, cell lysis buffer, luciferase and luciferase substrate

Equipment

Regular refrigerator, -80oC freezer, CO2 incubator, Biosafety cabinet type II, Centrifuge, Microcentrifuge, Luminometer

 


Procedure

Freezing, storage and reviving cells

The protocol described below is for HEK293T cells. We used same protocol for storing cell lines such as HepG2, HCT, HeLa, MEF, and mouse Leydig cells, in appropriate growth media for at least 2 years at -80oC. All steps involving cells and media are performed aseptically under a biosafety cabinet type II (tissue culture hood)

  1. Grow HEK293T cells to confluent in T25 or p60 plates in DMEM supplied with penicillin-streptomycin and 10% FBS or in appropriate growth media for other cell types.
  2. Wash cells in TCD once with 0.5 ml TE (Trypsin-EDTA), then incubate with 0.5 ml of TE for 5 min at 370C.
  3. Resuspend trypsinized cells with 0.5 mL in 6.5 ml media without serum using micropipette.
  4. Add 6.5 ml media without serum, mix by pipetting up and down, then centrifuge at 1500 rpm for 3 min.
  5. Remove supernatant and add 2.85 ml growth media containing serum, and resuspend cells using pipet.
  6. Add 0.15 ml DMSO and mix by swirling.
  7. Immediately transfer 1.0 ml into each freezing vial labeled with cell type, passage number, date etc.
  8. Transfer the tubes into a foam 15 mL-tube holder at -800C, and cover with saran wrap or with another foam tube holder upside down on it. 

Time Taken: 30 min

  1. When needed, thaw a cell vial taken out of -800C freezer with warm (~30oC) water in a small beaker. 
  2. Wipe dry water from outside the tube, and wipe once with a paper towel soaked with 70% ethanol.
  3. Pipet entire content into a p60 or T25 dish containing 10 ml growth media.
  4. Place the plate in a CO2 incubator (5% CO2, 370C) for 2 hours.
  5. Replace the media with fresh 5 ml growth media and return the dish to the CO2 incubator.

Time Taken: 2:30 h

Notes/Comments: 5% DMSO worked well for cells grown in media containing 10% serum. For cells grown in serum concentration, concentration of DMSO and /or serum may have to be adjusted. 

Reusing tissue culture dishes

The protocol below is only for cell maintenance/passage:

  1. Wash cells in TCD once with 0.5 ml TE (Trypsin-EDTA), then incubate with 0.5 ml of TE for 5 min at 370C.
  2. Add 0.5 ml of media without serum, and resuspend cells using micropipette.
  3. Transfer 0.1 ml into a fresh microfuge tube and add 1.0 ml media, mix by inverting.
  4. Microcentrifuge for 3 min at 3000 rpm.
  5. Discard remainder of the cells & media from the TC dish/plate. 

Notes/Comments: This splitting ratio (1:50) is used for maintenance of HEK 293T cells in T25 or p60 dishes. They become confluent within a week. For slow growing cells, we used 1:10 ratio. If needed, the remaining cells can be centrifuged to remove trypsin containing media and used for setting up experiments.

  1. Wash the TCD with 0.5 ml serum-free media by pipetting media all over cell-growing surface. Try to remove cells from the cell-growing surface. Discard the media.
  2. Repeat this washing of cell-growing surface once more. 
  3. Add growth media (4 ml to p60 or 5 ml to T25) to the washed dish.
  4. Discard supernatant media from the centrifuged tube. Resuspend in 0.5 ml growth media.
  5. Transfer 0.1 ml to the TCD and mix by swirling.
  6. Return the plate to the CO2 incubator.

Notes/Comments: It is important to remove cells completely from growing surface in the washing steps. Increase washing, if cells are not completely removed, otherwise newly plated cells will grow on top of the left-over cells, which will give a colony-like appearance. Washing is much easier in p60 plate than in T25 dishes as all surfaces are not accessible in T25. Colony-like appearance was observed after 4-5 passages in T25 plates compared to 6-7 passages in p60 plates. The colony-like appearances disappear if seeding is done into a new dish (T25 or p60). We also observed more cells (140%) are produced in p60 dishes compared to T25 over the same time, even though we used less growth media in p60 dishes (4 ml vs 5 ml).   

Time Taken: 45-60 min

Simplified protocol for calcium phosphate (CaPh) transfection

The procedure given is for 12-well plate.

  1. Add 500 ul of 2x Hepes solution in a 15 ml sterile tube.   
  2. Mix 60 ul of CaCl2 (2M) with 450 ul of H2O in a microfuge tube.
  3. Attach a 1-ml sterile pipet to an auto-pipetor and the other end to a pipetting 200ul tip. Insert the tip halfway into 2x Hepes taken in the 15 ml tube. Start bubbling by pressing the release button of the auto-pipetor.
  4. Using a micropipette (200 ml), add CaCl2-H2O dropwise to the bubbling 2x Hepes solution, until all solution were added. Continue bubbling for another 30 sec.
  5. Transfer appropriate volume (75 ml for 1-well, 150 ml for 2-well etc.) in 0.65 ml tube containing DNA in a total volume of 15 ml. 
  6. Mix the tubes by briefly vortexing and incubate up to 15 min at room temperature. The transfection efficiency gradually increases up to 15 min, then start to decrease as shown in Figure 1 below.
  7. In the hood, add the DNA-CaPh mixture dropwise to cells (50-70% confluent) in growth media plated overnight. No need to replace growth media with serum-free media.

Notes/CommentsFor duplicate or triplicate samples, pipet the mixture up-down 2-3 times before dispensing as it is a colloidal mixture. This will minimize variation among the replicas, especially for luciferase or beta-galactosidase assays. 

  1. When all wells are added with transfection mixture, swirl the plate gently to spread the mixture evenly.
  2. Return the plate to the incubator. 
  3. If desired, replace the media with conditioned media after 6-8 hours, or leave the cells in transfection media until harvest.

Time Taken: 20-30 min

As shown in the figure 2, this method (bubbling without DNA) has better transfection efficiency in four cell lines tested compared to when bubbling was done with individual DNA samples. The DNA used was CMV promoter in pGL3 vector expressing luciferase. It is possible bubbling with DNA may cause some damage or denaturation of the DNA.

Troubleshooting

If cells are not reviving upon storage 1)  during freezing, slow cool in a foam holder, 2) increase cell density in freezing vials, 3) increase growth media over two times during revival, and don’t exceed 3 hours of incubation as it still contains DMSO, or 4) adjust DMSO percentage. MEF cells require longer time to revive. 

If you get colony-like appearance when TCDs are reused for cell maintenance (usually appears after 4-6 reuse): 1) increase washing of the plate with media without serum, or 2) plate cells in a new plate.    

If transfection efficiency is very low (transfection efficiency is generally very low for HCT, HepG2, Leydig cells), use lipid-based transfection reagents. 

Figure 1. CMV promoter driven plasmids expressing fire-fly luciferase (0.3 microgram) and beta-galactosidase (0.3 microgram) incubated with 225 microliter of bubbled calcium phosphate reagent for 5- 30 min at room temperature before being added to HEK293T cells. After 40 h, the transfected cells were harvested and assayed for luciferase, beta-galactosidase and protein. The transfection efficiency is determined from normalized RLU value over beta-galactosidase and protein amount and is set at 1.0 in cells incubated for 5 min with the bubbled reagent. Each bar is the mean ± SE of a representative experiment performed in triplicate. 

Figure 2. CMV promoter driven plasmids expressing fire-fly luciferase (0.3 microgram) and beta-galactosidase (0.3 microgram) incubated with after bubbling (without DNA) or during bubbling (with DNA), then added to indicated cells. After 40 h, the transfected cells were harvested and assayed for luciferase, beta-galactosidase and protein. The transfection efficiency is determined from normalized RLU value over beta-galactosidase and protein amount and is set at 1.0 in cells transfected with bubbling DNA. Each bar is the mean ± SE of a representative experiment performed in triplicate. Transfection efficiency is increased in all cell types studied when the reagent was added after bubbling (Without DNA) compared to when bubbled with DNA (*p <0.005). 

 

 

 

 


Time Taken

Included in each section

Notes and Comments

Included in each section


References

Koller KJ, Whitehorn EA, Tate E, Rie, T, Aguilar B, Chernov-Rogan T, et al. (1997) A generic method for the production of cell lines expressing high levels of 7-transmembrane receptors. Anal. Biochem. 250, 51–60.

2. Chen C. and Okayama H. (1987) High-efficiency transformation of mammalian cells by plasmid DNA. Mol. Cell. Biol. 7, 2745–2752.

3. Maser, RL., Magenheimer BS., Zien CA., and Calvet JP. (2003) Transient Transfection Assays for Analysis of Signal Transduction in Renal Cells. Methods Mol Med 2003;86:205-17. doi: 10.1385/1-59259-392-5:205.

4. Calcium Phosphate Transfection Kit Most cost effective transfection reagent kit for transient and stable transfection of DNA into mammalian cells https://www.sigmaaldrich.com/US/en/product/sigma/caphos


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